PROJECT SUMMARY Early embryogenesis is the most vulnerable stage in the life cycle of mammals. Embryonic stem cells (ESCs) in the uterus have a dedicated task of rapid cell proliferation for the organismal development, but they can be adversely affected by many environmental factors, such as infection and inflammation. Currently, we know little about how ESCs cope with stress conditions. The interferon (IFN) system is the central part of antiviral innate immunity in differentiated somatic cells. We have recently demonstrated that ESCs are unable to express IFN, indicating that ESCs may not have a functional IFN-based antiviral mechanism. This finding raises important questions about the rationale for ESCs to not have such a critical defense mechanism and how do ESCs deal with viral infection. Using a synthetic dsRNA-based virus-free system, we have unexpectedly found that Dicer knockout ESCs (D-/-ESCs) seem to be able to express IFN? and are highly sensitive to the increased cytotoxic effects of dsRNA. Similarly, B2RNA, a cellular RNA derived from the transcripts of transposable elements, showed remarkably similar effects to these caused by dsRNA. These findings challenge our view that the lack of IFN response is intrinsic in ESCs. Instead, our finding suggests that the basic machinery of the IFN system is functional in ESCs, but it may normally be repressed by Dicer. It is known that cellular RNA with dsRNA structures, derived from injured cells, misprocessed RNA and transcripts of transposable elements, can elicit antiviral responses that cause cellular damage similar to viral infection. We hypothesize that, by repressing IFN response, Dicer may act as a sentinel enzyme to prevent excessive antiviral responses that could potentially harm ESCs during early embryogenesis. Using mouse ESCs as a model system, we will test this hypothesis by: 1) using synthetic dsRNA as viral RNA mimics to determine the functionality of the major signaling events essential for the IFN system in D-/-ESCs (Aim 1), 2) using B2RNA as a physiologically relevant cellular RNA to investigate the activation of the IFN system and determine the role of Dicer in restricting B2RNA-induced cytotoxicity (Aim 2), and 3) reconstituting D-/-ESCs with ectopically expressed Dicer from both a plasmid vector and from synthetic Dicer-mRNA. The reconstituted ESCs will be used to validate the results obtained from dsRNA and B2RNA in D-/-ESCs. The successful completion of the proposed project is expected to provide significant insight into the unique immune properties of ESCs, specifically on how the IFN-based antiviral innate immunity is delicately controlled by Dicer to ensure normal growth and development at the early stage of organismal development.